Lyme disease, human anaplasmosis, human babesiosis, ehrlichiosis, and Powassan virus are increasingly prevalent tick-borne infections in the US. The behavior of the black-legged tick, Ixodes scapularis, is an important mediator of the risk of human infection. In a small pilot study we found that significant larval and nymphal questing occurred nocturnally and at times when diurnal questing was absent. Nocturnal questing concentrates tick activity to periods when important hosts for immature I. scapularis are most active. These animals are reservoirs of B. burgdorferi, A. phagocytophilum, Ba. microti and/or Ehrlichia muris and nocturnal questing activity of I. scapularis larvae likely results in a greater percentage of larvae feeding on disease competent reservoirs resulting in a greater percentage of infected questing nymphs. We propose to dissect the temporal patterns of nighttime questing to examine impacts on tick-borne disease ecology. We will test the following hypotheses: 1) the frequency of daytime vs nighttime questing will vary throughout a season or by geographic location, with shifts to nighttime questing occurring during hotter and drier periods or locations; 2) Tick body burdens on nocturnally active small mammals will be relatively higher during hotter and drier periods or locations in comparison with animals active during the day. This could drive higher pathogen prevalence in locations with greater nighttime questing; 3) Infected nymphs behave differently than uninfected nymphs. Infection likely carries an energetic cost and infected ticks may adjust questing behavior to periods of lower environmental stress. We will determine whether or not infection with any of the tick borne pathogens, (B. burgdorferi, A. phagocytophilum, Ba. microti or Ehrlichia muris) is correlated with the nocturnal questing behavior of I. scapularis nymphal ticks. Successful completion of these aims will provide important information that will greatly facilitate future analyses the impacts of environmental change on disease ecology and the risk of tick-borne disease.